Kammerer Bernd, Frickenschmidt Antje, Gleiter Christoph H, Laufer Stefan, Liebich Hartmut
Institute of Pharmacology and Toxicology, Division of Clinical Pharmacology, University Hospital Tübingen, Otfried-Müller Strasse 45, D-72076 Tübingen, Germany.
J Am Soc Mass Spectrom. 2005 Jun;16(6):940-7. doi: 10.1016/j.jasms.2005.02.018. Epub 2005 Apr 20.
As RNA turnover seems to be impaired in cancer patients, modified nucleosides have been evaluated as potential tumor markers. Modified nucleosides are mainly formed post-transcriptionally in tRNA, set free during RNA metabolism, and excreted in urine. Especially methylated nucleosides play an important role, as their levels are higher in urine from cancer patients. For structural elucidation of known and unknown nucleosides from urine samples of cancer patients, MALDI-TOF MS and MALDI-PSD were used for the first time. This technique generally ensures high sensitivity, mass resolution, and accuracy. In our analytical approach we prepurified nucleosides from urine by affinity chromatography and subsequently separated them by semipreparative high performance liquid chromatography. The different fractions were collected separately and analyzed by MALDI-TOF MS and PSD-MALDI using a mixture of six low molecular weight calibrants for internal or external calibration. The molecular totals formulas based on a mass accuracy of 10 ppm and below were calculated and a systematic data base search was performed. The inherent problem of the MALDI-technique, the reduced sensitivity for low molecular weight substances caused by matrix suppression effects, was reduced by our technique. We identified several nucleosides in urine, which were previously identified via retention times and UV spectra of standards after HPLC analysis. Eight further nucleosides were observed. This work demonstrates for the first time the potential of MALDI-TOF and PSD-MALDI in combination with semipreparative HPLC for assignment of nucleosides in urine. The particularly high mass accuracy of this mass spectrometric method provides opportunities for identifying unknown compounds.
由于癌症患者的RNA周转似乎受损，修饰核苷已被评估为潜在的肿瘤标志物。修饰核苷主要在转录后于tRNA中形成，在RNA代谢过程中释放，并随尿液排出。特别是甲基化核苷发挥着重要作用，因为它们在癌症患者尿液中的水平较高。首次使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和基质辅助激光解吸电离后源衰变（MALDI-PSD）对癌症患者尿液样本中已知和未知核苷进行结构解析。该技术通常可确保高灵敏度、质量分辨率和准确性。在我们的分析方法中，我们通过亲和色谱法对尿液中的核苷进行预纯化，随后通过半制备高效液相色谱法对其进行分离。分别收集不同的馏分，并使用六种低分子量校准物的混合物进行内部或外部校准，通过MALDI-TOF MS和PSD-MALDI进行分析。基于10 ppm及以下的质量准确度计算分子总式，并进行系统的数据库搜索。我们的技术降低了MALDI技术固有的问题，即基质抑制效应导致的对低分子量物质灵敏度降低的问题。我们在尿液中鉴定出了几种核苷，这些核苷之前是通过高效液相色谱分析后的标准品保留时间和紫外光谱鉴定出来的。还观察到另外八种核苷。这项工作首次证明了MALDI-TOF和PSD-MALDI与半制备高效液相色谱相结合在鉴定尿液中核苷方面的潜力。这种质谱方法特别高的质量准确度为鉴定未知化合物提供了机会。
